comparative karyological analysis of three diplozoon ... · three species are characterized by the...

Sci Parasitol 16(3):71-82, September 2015 ISSN 1582-1366

ORIGINAL RESEARCH ARTICLE

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Comparative Karyological analysis of three Diplozoon Species

(Monogenea, Diplozoidae), Gill Parasites of Schizothorax species –

first report from the Kashmir Valley, India

Fayaz Ahmad1, Tanveer A. Sofi1, Khalid Majid Fazili2, Bashir A. Sheikh1,

Omer Mohi ud Din Sofi3, Kamal Jaswal4

1 – Department of Zoology, University of Kashmir, Srinagar – 190006.

2 – Department of Biotechnology, University of Kashmir, Srinagar.

3 – SK University of Agricultural Sciences and Technology, Shuhama, Aluestang.

4 – Department of Applied Animal Sciences, Dr. B. R. Ambedkar Central University, Lucknow, U.P.

Correspondence: Tel. +919797127214, E-mail [email protected]

Abstract. The present study has revealed new data on chromosome complements of diplozoid parasites, namely

Diplozoon Kashmirensis Kaw, 1950 from Schizothorax esocinus; Diplozoon aegyptensis Fischthal and Kuntz, 1963

from Schizothorax plagiostomum; Diplozoon guptai Fayaz and Chishti, 2000 from Schizothorax curvifrons. All the

three species are characterized by the same number of chromosomes i.e., 2n=14 in which D. kashmirensis is

characterized by seven pairs of long (up to 14.13 μm) chromosomes and all chromosome pairs are acrocentric.

Karyotype of D. aegyptensis also contains 14 chromosomes but with different morphology in which first six

chromosomes are metacentric and last eight chromosomes are acrocentric and the is length ranging up to 9.78

µm. Chromosomes of D. guptai ranges from 5.39 μm to 8.02 μm in which first four chromosomes are metacentric,

two chromosomes are submetacentric, two chromosomes are subtelocentric and last six chromosomes are

acrocentric. The present study describes for the first time the chromosome structure and number of three

Diplozoon species from the host Schizothorax species of Dal lake of Kashmir Valley.

Keywords: Diplozoon; Schizothorax; Karyotype; Kashmir; Metacentric; Acrocentric; Telocentric; Subtelocentric.

Received 22/04/2015. Accepted 21/06/2015.

Introduction Helwig (1958) stated that “each species has its own chromosomal integration which is as characteristic and diagnostic for it as any external morphological trait”. The karyological

studies shows differences in the chromosomal number and chromosomal morphology which frequently distinguish one species from its relatives which are not obvious at the morphological level. Thus, cytogenetical information about the species is essential for the



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modern systematic, since the species are said to be the objective reality of some particular genetic continuity. Cytogenetics – the analysis of diploid numbers, karyotypes, metrical data of metaphase chromosomes, existence of specialized chromosomes, behaviour, and arrangement of nuclear bodies are the means by which specific homology as well as analogy between the allied species and groups could be derived. The nuclear cytogenetics may also provide an independent set of data which can help in tracing the more reliable, evolutionary pathways of different animal groups. Diplozoid monogeneans are gill ectoparasites of freshwater, mainly cyprinid fish are represented by two dozens of species in Europe (Khotenovsky, 1985). Systematics of the family remains problematic due to a relatively high interspecific similarity in morphological features and limited number of species included in molecular comparisons (Matejusova et al., 2001; 2004; Gao et al., 2007; Civanova et al., 2013; Avenant-Oldewage et al., 2014). Therefore, supplementary approaches would help in the assessment of species delimitation and/or phylogeny. To date, only few studies have been focused on diplozoid cytotaxonomy and none from the Kashmir valley. Koroleva (1968a, 1968b; 1969) showed chromosome morphology of six species from various fish hosts, and Koskova et al. (2011) showed specified cytogenetic characteristics of four of them. Species of Paradiplozoon bliccae, Paradiplozoon sapae, Paradiplozoon nagibinae, Paradiplozoon pavlovskii and Paradiplozoon homoion has 14 acrocentric elements in their diploid set (2n=14), while Diplozoon paradoxum has 2n=8. Baer and Euzet (1961); Bovet (1967); Koroleva (1969) showed that three undetermined diplozoids showed either 14 or 10 chromosomes in diploid set and Koroleva (1968b) studied two other species Eudiplozoon nipponicum and Paradiplozoon megan, revealed n=7 on the basis of meiotic bivalents without any information on the chromosome morphology. The present study describes for the first time the chromosome structure and number of three Diplozoon spp. from Kashmir Valley. The three species of Diplozoon, parasitizing Schizothorax species, have been the objects of our cytogenetic study, aimed at a comparison of a structure of their

chromosome sets and an analysis of hypothetic routes of karyotype evolution within the group. Material and methods Parasite material Ten to fifteen fishes per survey were collected using gill nets consisting of four sections with varying mesh sizes of 90, 110 and 130 mm, respectively. The fishes were identified based on the size of the snout as suggested by Skelton (2001). Parasitic specimens after collected from the fish hosts were then identified using reference keys of Yamaguti (1971), Bauer (1987), Chubb et al. (1987) and Hoffman (1999). In addition, local literature was also used during the identification process. Adults of three monogenean species were obtained from gills of three specific fish hosts: a total of 27 specimens of D. kashmirensis were collected from 19 freshwater Schizothorax esocinus; 35 specimens of D. aegyptensis were collected from 12 Schizothorax plagiostomum and 12 specimens of D. guptai from 14 Schizothorax curvifrons from the Dal Lake of Kashmir, Srinagar India. Collections were made during the years 2013 and 2014. For species determination, haptors of each individual were cut off, mounted on a slide in ammonium picrate– glycerin mixture (Ergens, 1969), and identified according to morphometry of the attachment apparatus (clamps and central hooks), using an Olympus BX 50 light microscope equipped with a differential interference contrast (Nomarski DIC). Chromosome preparations The middle part of each individual containing testes and ovary was used for spreading analysis according to Frydrychova and Marec (2002). Parasite tissue was placed into hypotonic solution (0.075 M KCl) for 15–20 min and then put into freshly prepared Carnoy fixative (ethanol/chloroform/acetic acid, 6:3:1) for 20–30 min. Fixed tissue pieces were transferred into a drop of 60% acetic acid on a slide and thorn by tungsten needles making cell suspension. It was overspread along the slide on a heating plate (45°C). Slides were dehydrated in an ethanol series (70%, 80%, 100%, 1 min each) and stored at −20°C until further use.



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Karyological analysis ● Pre-treatment The testes, ovaries and the uterus, as the case may be, were transferred directly to the hypotonic solution. In order to get good spreading of the chromosomes the various hypotonic solutions listed below were tried:

a. 0.9% sodium citrate solution for 15 min. at room temperature (Ford and Hamerton, 1956).

b. 0.7% sodium citrate solution for 15 min. at room temperature (Eaker et al., 2001).

c. Simple distilled water at 37°C for 15-20 min.

d. Double-distilled water at 37°C for 15-20 min. (Makino and Nishimura, 1952; Spriggs et al., 1962).

e. An aqueous solution of 0.4% and 0.5% sodium chloride for 20-25 min. (Taylor et al., 1957).

Out of all these, 0.9% sodium citrate solution for 15-20 min. at 37°C gave optimum spreading. In a few cases, the material was treated with 0.1% colchicine solution for 1h at room temperature to arrest the metaphase plates. However no useable preparation was obtained by this treatment. ● Preparation of the slides Usually the material was stained and squashed immediately after fixation, but whenever needed, it was preserved in 70% alcohol for 1-2 days. The fixed tissue was processed according to the different staining methods used for the study of chromosomes. Their details are given below: a) Gomori’s haematoxylin squash technique

(Belling, 1926) The fixed parasites were hydrolysed in 1N HCl for 10-12 min. at 60°C and were washed in distilled water in order to remove all traces of

HCl. The material was then kept in filtered Gomeri’s stain for about 1h at 60°C. The stained tissue was differentiated in 45% acetic acid for 15-20 minutes at 60°C. The material was then squashed in a drop of 45% acetic acid on a clean slide, by placing gently a cover slip over it and then applying a gentle but firm pressure through the folds of a blotting paper. The slides thus prepared, were stored for the night in the freezing chamber at -10°C followed by Melander and Wingstrand (1953). The temporary preparations were made permanent by separating the cover slips from the slide with the help of a sharp blades and dehydrating them in rectified and absolute alcohol for 10-20 min. each. Finally, after mounting them in Euprol, the slides were placed in an oven at 60°C for 2-3 days for drying. The used technique was not efficient enough. In most of the cells, even a gentle pressure during squashing resulted in damaging the cells and thus the constancy of the chromosome number was disturbed. Furthermore, a good spreading of the small chromosome could not be obtained by this technique. b) Air drying method with carbol-fuchsin

staining A slightly modified technique of Crozier (1969) was followed. The tissue after fixation with Carnoy’s fixative was placed on a clean slide in a drop of 60% acetic acid. If the tissue did not dissociate rapidly, a gentle tapping with the help of a forceps was given. A few drops of the fixative were added to the preparation and the slide was tilted in all directions so as to ensure the maximum spreading of the cells. These air–dried slides were then stained in Carbol-fuchsin for the night, differentiated by giving a dip in 95% alcohol and dehydrating in n-butyl alcohol for 10 min. Slides were finally mounted in euparol. This technique yielded the best results and was, therefore, applied exclusively. This procedure has an advantage as the air dried slides can be kept indefinitely and stained at any convenient time. Further, for outstation collection, this is the most suitable process as Gomori’s haematoxylin squash method requires



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laboratory equipment such as an oven, refrigerator. The slides were Giemsa stained. Slight variation in the time duration of a particular step in processing the material for the chromosome preparations were enforced, off and on, whenever required. Mean absolute and relative lengths as well as centromeric indices were calculated in ten (D. kashmirensis, D. agyptensis, and D. guptai) best mitotic spreads. The terminology relating to centromere position follows that of Levan et al. (1964). A chromosome is metacentric (m) if the ci falls in the range of 37.5–50.0, submetacentric (sm) if 25.0–37.5, subtelocentric (st) if 12.5–25.0; acrocentric (a) if ci<12.5 and telocentric if ci=0. When the centromere position was on the borderline between two categories, both are listed. Statistical methods Parametric as well as the non-parametric tests were used for analyzing relative length of chromosomes. A computer program (Minitab for windows) was used for data analysis. Chi-Square analysis was used to see the statistical significance in chromosome lengths. Pearson’s correlation was used to find correlation between different species of helminths. Student’s t-test was used to test the differences which were considered to be significant when the p-value obtained was less than 0.05.

Results Diplozoon kashmirensis Kaw, 1950 Analysis of mitotic metaphase spreads from ten specimens of Diplozoon kashmirensis showed that the karyotype of D. kashmirensis comprised 14 acrocentric chromosomes (2n=14; figure 1a). The karyotype formula can be summarized as 2n=14a (figure 1b). The longest pair is 14.13 μm and the shortest pair is 6.21 μm long (table 1), the fundamental arm number (NF) = 7 and total chromosome length (TCL) is 72.57 μm. Arm ratio of the complement ranges between 7.67-14.15 and the centromeric index ranges 6.60 to 12.28. Chromosome pair no. 2, 3 and 4 are nearly similar in their size and are very difficult to identify on the basis of chromosome morphology (Student T-test; P-value = 0.002; P<0.05), precise identification of second, third and fourth chromosome pairs is rather difficult because of the low degree of significance of length differences, but there is significant difference between chromosome pairs of 5, 6 and 7 (P-Value = 0.000; P<0.001; Student T-test). Whereas statistical processing of the established relative lengths of chromosome pairs of 1 & 2 (P>0.05) and 4, 5, 6 and 7 (P<0.005) are significant due to length difference between them. So, on the basis of absolute length and centromeric position, the chromosomes have been arranged in order of decreasing length in an ideogram (figures 2a, 2b).

Karyotype Formula: (K) 2n = 14 = 14a

Figure 1 (a) Chromosome preparation of Diplozoon kashmirensis; (b) Ideogram constructed from mitotic cells of Diplozoon kashmirensis stained with Giemsa (a = acrocentric)

1 2 3 4 5 6 7

Scale Bar 10µm

a

b



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Figure 2 (a) Idiogram of Diplozoon kashmirensis; (b) Error Bars with Standard Deviation of Diplozoon kashmirensis

Table 1. Measurements and classification of chromosomes of Diplozoon kashmirensis Kaw, 1950

Chromo-some pair

number

Length of short arm

(µm) ‘S’

Length of long arm (µm) ‘L’

Total Length/Absolute Length (µm) L+S

Arm Ratio (L/S)

Relative Length

(%)

Centro-meric

Index (ci) Classification

1 1.63 12.5 14.13 7.67 19.47 11.54 Acrocentric T-Value = -25.39 P-Value = 0.002 P<0.05

2 1.47 10.5 11.97 7.14 16.49 12.28 Acrocentric

3 1.22 9.80 11.02 8.03 15.19 11.07 Acrocentric

4 1.12 9.00 10.92 8.03 15.05 10.26 Acrocentric 5 0.89 8.80 9.69 9.89 13.35 9.19 Acrocentric T-Value = -10.88

P-Value = 0.000 P<0.001

6 0.63 8.00 8.63 12.60 11.89 7.30 Acrocentric 7 0.41 5.80 6.21 14.15 8.56 6.60 Acrocentric

Diplozoon aegyptensis Fischthal et Kuntz, 1963 The somatic complement of Diplozoon aegyptensis species revealed a diploid number of 2n = 14 (figure 3a) comprising first three

pairs of chromosomes as metacentric and last four pairs of chromosomes as acrocentric in which a fundamental arm number (FN) equals 10 (figure 3b). The chromosomes range in length between 7.11 µm to 8.08 µm. The total

a

b



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length of the haploid complement equals 55.78 µm. Arm ratio of the complement ranges between 1.09–17.23 and the centromeric index ranges between 5.49–47.90 (table 2). On the basis of total length of chromosomes and relative length there is less significant difference between first three pairs of metacentric chromosomes (P=0.002; P<0.05;

Student T-test) and significant difference between last four pairs of acrocentric chromosome pairs (P=0.001; P<0.001; Student T-test). The absolute length and centromeric position of the chromosomes have been arranged in order of decreasing length in an ideogram (figures 4a, 4b).

Karyotype Formula: (K) 2n = 14 = 6m + 8a

Figure 3 (a) Chromosome preparation of Diplozoon aegyptensis; (b) Karyotype constructed from mitotic cells of Diplozoon aegyptensis stained with Giemsa (m = metacentric; a = acrocentric;)

1 2 3 4 5 6 7

Scale Bar 10µm

a

b

a



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Figure 4 (a) Ideogram of Diplozoon aegyptensis; (b) Error Bars with Standard Deviation of Diplozoon aegyptensis

Table 2. Measurements and classification of chromosomes of Diplozoon aegyptensis Fischthal et Kuntz, 1963

Chromo-some pair

number

Length of short arm

(µm) ‘S’


Total Length/Absolute Length (µm) L+S

Arm Ratio (L/S)

Relative Length

(%)

Centro-meric

Index (ci) Classification

1 3.87 4.21 8.08 1.09 14.49 47.90 Metacentric T-Value = -20.56 P-Value = 0.002 P<0.05

2 3.31 4.07 7.38 1.23 13.23 44.85 Metacentric 3 3.03 3.81 6.84 1.26 12.26 44.30 Metacentric

4 0.93 8.85 9.78 9.52 17.53 9.51 Acrocentric T-Value = -14.82 P-Value = 0.001 P<0.001

5 0.77 7.89 8.66 10.25 15.53 8.89 Acrocentric 6 0.51 7.42 7.93 14.55 14.22 6.43 Acrocentric 7 0.39 6.72 7.11 17.23 12.75 5.49 Acrocentric

Diplozoon guptai Fayaz and Chishti, 2000 Diploid chromosome number of D. guptai is 2n=14 as revealed after examination of mitotic metaphase spreads from 13 specimens (figure 5a). Karyotype (figure 5b) included two metacentric (nos. 1 and 2); one submetacentric (no. 3); one subtelocentric (no. 4) and three acrocentric (nos. 5, 6 and 7) chromosome pair; the karyotype formula may be summarized as 2n=4m+2sm+2st+6a. The chromosomes are comparatively large; the smallest and the largest chromosomes measured 5.39 μm and 8.02 μm, respectively (for chromosome measurements, see table 3). The number of chromosome arms (NF) is 20 and total

chromosome length (TCL) is 47.25 μm. Arm ratio of the complement ranges between 1.04–34.53 and the centromeric index ranges between 2.81–49.00 (table 3). Length difference between first three pairs are much less and there is less significant difference between them (P=0.002; P<0.05; Student T-test), and these are classified metacentric and submetacentric pairs, but there are significant length difference between four pairs of chromosomes (P=0.001 ; P<0.001 ; Student T-test). On the basis of absolute length and centromeric position, the chromosomes have been arranged in order of decreasing length in an ideogram (figures 6a, 6b).

b



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Karyotype Formula: (K) 2n = 14 = 4m + 2sm + 2st + 6a

Figure 5 (a) Chromosome preparation of Diplozoon guptai; (b) Karyotype constructed from mitotic cells of Diplozoon guptai

stained with Giemsa (m = metacentric; sm = Submetacentric; st = Subtelocentric; a = acrocentric)

Figure 6 (a) Ideogram of Diplozoon guptai; (b) Error Bars with Standard Deviation Diplozoon guptai

1 2 3 4 5 6 7

a

b

Scale Bar 10µm

a

b



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Table 3. Measurements and classification of chromosomes of Diplozoon guptai Fayaz and Chishti, 2000

Chromo-some pair

number

Length of short arm

(µm) ‘S’


Total Length/ Absolute

Length (µm) L+S

Arm Ratio (L/S)

Relative Length

(%)

Centro-meric Index

(ci)

Classification

1 3.43 3.57 7.00 1.04 14.81 49.00 Metacentric T-Value = -23.32 P-Value = 0.002 P<0.05

2 3.13 3.45 6.58 1.10 13.93 47.57 Metacentric 3 2.12 3.91 6.03 1.84 12.76 35.16 Submeta-

centric 4 0.93 4.46 5.39 4.80 11.41 17.25 Subtelo-

centric T-Value = -12.16 P-Value = 0.001 P<0.001 5 0.51 7.51 8.02 14.73 16.97 6.36 Acrocentric

6 0.37 7.11 7.48 19.22 15.83 4.95 Acrocentric 7 0.19 6.56 6.75 34.53 14.29 2.81 Acrocentric

Table 4. Comparative chromosomes number and morphology of Diplozoon species

Family species Chromosome no. and morphology Authority

Diplozoon paradoxum 2n=8 (3m+1a) Koskova et al. (2011) Paradiplozoon bliccae 2n=14 (7a) Koskova et al. (2011) Paradiplozoon sapae 2n=14 (7a) Koskova et al. (2011) Paradiplozoon nagibinae 2n=14 (7a) Koskova et al. (2011) Diplozoon kashmirensis (Kaw, 1950) 2n=14 (14a) Present study Diplozoon aegyptensis (Fischthal et Kuntz, 1963)

2n=14 (6m+8a) Present study

Diplozoon guptai (Fayaz and Chishti, 2000) 2n=14 (4m+2sm+2st+6a) Present study Eudiplozoon nipponicum 2n=7 Koroleva (1968b) Paradiplozoon megan 2n=7 Koroleva (1968b) Diplozoon paradoxum 2n=8, (3m+1a) Koroleva (1968a,b) Paradiplozoon bliccae (syn. Diplozoon gussevi) 2n=14, (7a) Koroleva (1968a,b)

Paradiplozoon bliccae (syn. Diplozoon markevitchi)

2n=14, (7a) Koroleva (1968b, 1969)

Paradiplozoon sapae 2n=14, (7a) Koroleva (1969) Paradiplozoon nagibinae 2n=14, (7a) Koroleva (1969) Paradiplozoon pavlovskii 2n=14, (7a) Koroleva (1968a,b) Paradiplozoon homoion 2n=14, (7a) Koroleva (1968a,b) Diplozoidae sp. 2n=14, (7a) Bovet (1967) Diplozoidae sp. (sp. n.) 2n=10, (2m+3a) Koroleva (1969) Diplozoidae sp. 2n=7 Baer & Euzet (1961) Diplozoidae sp.

2n=7 Bovet (1967) Incorrect data according to Koroleva (1968b)

Table 5. Comparative Relative Lengths (%) of Monogenean Trematodes

Chromosome Pair No. Diplozoon kashmirensis

Kaw, 1950 Diplozoon aegyptensis

Fischthal et Kuntz, 1963 Diplozoon guptai

Fayaz and Chishti, 2000

1 19.47 14.49 14.81 2 16.49 13.23 13.93 3 15.19 12.26 12.76 4 15.05 17.53 11.41 5 13.35 15.53 16.97 6 11.89 14.22 15.83 7 8.56 12.75 14.29

Discussion Karyological characterization of Monogenea species is a neglected subject. However, the study of karyotype of Diplozoon spp. was the

first study in Kashmir Valley. Regarding diplozoids, 17 species have been studied cytogenetically to date (13 identified and 4 unclassified taxa, table 4); karyotypes of only 9 additional monogeneans have been published



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(Benazzi and Benazzi Lentati, 1976; Harris, 1985; Rohde, 1994; Cable and Harris, 2002). As summarized in table 4, all diplozoid species have chromosome sets comprising 14 acrocentric elements except two species which comprising 3 metacentric and 1 acrocentric elements. Taking into account a hypothesis that less advanced species of a group often have non-symmetric karyotypes (White, 1973), this karyotype seems to represent an ancestral type (Koroleva, 1969). Thus, Koroleva (1969) suggested that species with chromosome numbers lower than 14 might originate via Robertsonian centric-fusion translocations during evolution. Four analyzed karyotypes of D. paradoxum, P. bliccae, P. nagibinae, and P. sapae were previously studied by Koroleva (1968a; 1968b; 1969), and the data on number and classification of chromosomes fit well with the present results. However, our study has revealed new information on chromosome measurements of Diplozoon species of the Kashmir Valley. Koroleva (1968a; 1969) showed no interspecific differences among species with 2n=14. She reported maximum chromosome length from 3 to 5 μm in P. bliccae (syn. Diplozoon gussevi) and from 4 to 13 μm in D. paradoxum (Koroleva, 1968a). Our analysis revealed lower chromosome length, but such differences are likely related to different methodology used; it is known that air-dry and spreading techniques produce longer chromosomes than formerly used squashes (Reblanova et al., 2010). The most related congeners P. bliccae, P. nagibinae, and P. sapae (Matejusova et al., 2001, 2004; Gao et al., 2007) have equal number of 14 chromosomes of very similar morphology, all being acrocentric. However, our study showed that all the three species of Diplozoon examined contain 14 chromosomes with varying length of short and long arm and having different chromosome morphology. D. kashmirensis contains 14 chromosomes of which all are acrocentric (2n=14=14a) and have chromosome length ranging between 6.21-14.13 µm where as D. aegyptensis also contains 14 chromosomes but with different chromosome morphology in which the first three pairs are metacentric and rest of four pairs are acrocentric (2n=14=6m+8a) and have smallest and largest

chromosome length between 6.84 and 9.78 µm. The third species D. guptai differs markedly in chromosome morphology (2n=14=4m+2sm +2st+6a) but it has nearly the same chromosome length as of D. aegyptensis i.e., having a short and long arm between 5.39 and 8.02 µm. These data correspond well with the above-mentioned hypothesis of Koroleva regarding an evolution of the D. paradoxum karyotype from an ancestral type with seven one-armed pairs. Regarding interspecific differences of different species of Diplozoon spp. they show variation of their relative lengths (table 5). Thus, when comparing the relative length of Diplozoon kashmirensis with those of Diplozoon aegyptensis, the differences are not significant (T-value = -0.00, P-value = 0.999 and Pearson correlation = 0.190; P-value = 0.683; P>0.05). Those of Diplozoon kashmirensis with Diplozoon guptai (T-Value = 0.00, P-Value = 1.000 and Pearson correlation = -0.202, P-value = 0.664; P>0.05) again the differences are not significant and in Diplozoon aegyptensis compared to Diplozoon guptai (T-value = 0.00, P-Value = 0.999 and Pearson correlation= -0.127 P-Value = 0.787; P>0.05, here we again see that differences are not significant statically. Therefore, the noted differences in the relative chromosome lengths between the individual Diplozoon species cannot be used as a reliable criterion for establishing identification of the Diplozoon species. So, on the basis of centromeric index Diplozoon species can be used for the identification of different species. Thus, pericentromeric heterochromatin, occurring in acrocentric chromosomes of any of studied species with 2n=14, might be lost in the process of centric fusions. It is evident that further detailed cytogenetic study of subsequent diplozoid monogeneans will better reveal general routs of chromosome evolution within the relatively narrow group of interesting fish parasites. Acknowledgment The authors extend their thanks to the authorities of the Department of Zoology, University of Kashmir for the facilities provided. TAS is also highly thankful to Prof.



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Fayaz Ahmad for giving valuable suggestions while compiling this paper.

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comparative karyological analysis of three diplozoon ... · three species are characterized by the...

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